SOME  LYSINE  DERIVATIVES 


BY 

WILBUR  ARTHUR  LAZIER 


THESIS 


FOR  THE 


DEGREE  OF  BACHELOR  OF  SCIENCE 


CHEMISTRY 


COLLEGE  OF  LIBERAL  ARTS  AND  SCIENCES 


UNIVERSITY  OF  ILLINOIS 


1922 


■ 


/ 3 22 
L45 


UNIVERSITY  OF  ILLINOIS 


May— 24^. 192H— 


THIS  IS  TO  CERTIFY  THAT  THE  THESIS  PREPARED  UNDER  MY  SUPERVISION  BY 


-liibar- Yir-thu-r — Lazie  r- 


entitled Some-  Lya  ine— Dari-Ya.ti.Ye-S. 


IS  APPROVED  BY  ME  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF Ba cli S IQ IL _ Qj—  S_Q ie. EU2.0 lil _ CJi £J0 i&  t Xy 



Instructor  in  Charge 

Approved  : {jd  _ 

HEAD  OF  DEPARTMENT  OF 


Digitized  by  the  Internet  Archive 
in  2015 


https://archive.org/details/somelysinederivaOOIazi 


ACEUO’kVLBDGMEITT 

The  writer  wishes  to  express  to  Dr.  G.  5.  Marvel,  under 
whose  direction  this  work  was  done,  his  sincere  appreciation  of 
the  many  helpful  suggestions  tendered  during  the  course  of  the 
investigation. 


•• 


' 


TABLE  03?  CONTENTS 


Page 

I .  Ac  knowl e dgement 

II.  Table  of  Contents 

III.  Introduction  2 

IV.  Historical  and  Theoretical  4 

V.  Experimental 

Benzoyl  piperidine  9 

e-3enzoyl  amino  amyl  chloride  12 

6-Benz oyl  amino  amyl  cyanide  14 

6-Amino  caproic  acid  15 

6-Benzoyl  amino  caproic  acid  17 

6-Benzoyl  amino-a-brora  caproic  acid  18 

6-Benzoyl  lysine  20 

dl-Lysine  dihydrochloride  20 

Calcium  salt  of  6-benzoyl  amino-(X-hy- 

droxy  caproic  acid  21 

C-Amino- cx-hydroxy  caproic  acid  21 

Copper  salt  of  ex-hydroxy  caproic  acid  23 

o (-Hydroxy  caproic  acid  23 

Attempt  to  prepare  e-hydroxy  caproic  acid  24 

VI.  Summary  25 

VII.  Bibliography  26 


-2- 


INTRODUCTION 

For  a long  time  it  has  been  known  that  lysine  is  neces- 
sary in  the  diet  of  a normal  white  rat  in  order  to  produce  growth* 
Oshorne  & Mendelx  have  shown  that  gliadin  with  its  low  content  of 
lysine  as  the  sole  source  of  protein  of  the  diet  does  not  permit 

p 

normal  growth  unless  supplemented  "by  lysine.  They^  have  also  shown 
that  chickens  require  a sufficient  amount  of  lysine  to  make  normal 

growth. 

little  is  known  of  the  possibility  of  amino  acid  synthe- 
sis in  the  animal  organism.  Perfusion  experiments  with  the  sur- 
viving liver^  have  shown  that  (X-hydroxy  and  cx-keto  acids  may  he 
converted  to  the  corresponding  optically  active  amino  acids  exist- 
ing in  the  protein  molecule.  However,  in  the  living  organism,  the 

synthesis  of  a-amino  acids  by  the  reaction  of  NH_  and  non-nitrog- 

3 

A 

enous  bodies  has  not  been  proved.  Hart,  Nelson,  and  Pitz^  were 
unable  to  demonstrate  a synthesis  of  lysine  by  the  mammary  gland 
of  the  white  rat,  to  supply  an  adequate  milk  for  the  young. 

It  seems  probable  that  by  conducting  feeding  experiments 
with  compounds  containing  hydroxyl  groups  corresponding  to  the 
amino  groups  of  lysine  in  all  possible  combinations,  much  light 
might  be  shed  upon  the  possibility  of  lysine  synthesis  in  the  an- 
imal organism.  Such  experiments  have  never  been  conducted,  due 
to  the  necessary  compounds  being  unavailable. 

Therefore  this  investigation  was  carried  out  with  the 
idea  of  making  available  such  necessary  compounds  which  are  listed 
as  follows: 


-3- 


ot-£-diamino  caproic  acid  (Lysine) 
<x-amino  caproic  acid  (Nor-leucine) 
e-amino  caproic  acid  (e-leucine) 
e -amino -a-hy dr oxy  caproic  acid 
c(-amino-e-hydroxy  caproic  acid 
ot-e-dihydroxy  caproic  acid 
ct-hydroxy  caproic  acid 


NH  -CH-CH-CH-CH  -CH-COOH 
2 2 2 2 ° 


NH, 


CH  -CH  -CHo-CHo-CH-C00H 

3 2 2 2 Ah 

NH  -CH-CH-CH-CH  -CHp-C00H 
2 2 2 2 2 


NH  -CH  -CH  -CH  -CH  -CH-COOH 
2 2 ° ° ° - - 


2 2 


OH 


HO-CH  -CH  -CH  -CH  -CH-COOH 

2 2 2 2 T\ra 

2 

HO-CH  -CH0-CHo-CH9 -CH-COOH 
2 2 2 2 


CH„-CHo-CHo-CH9-CH-C00H 

2 2 c fog 


HO-CH  -CH  -CH  -CH  -CH  -COOH 
2 2 <2  6 2 


6-hydroxy  caproic  acid 

Determinations  of  free  amino  nitrogen  point  toward  the 
fact  that  in  lysine  only  one  of  the  amino  groups  is  combined  in 
peptide  synthesis.  The  e-group  is  commonly  thought  to  he  the  un- 

5 

combined  group.  Nor-leucine  has  been  shorn  by  Lewis  and  Root  to 
be  unable  to  supply  the  deficiency  of  a gliadin  diet  as  does  lysine 
and  shall  be  excluded  from  consideration  here. 


i. 


-4- 


HISTORICAL  AIID  THEORETICAL 


Lysine  was  discovered  in  1899  by  E.  Drechsel0  who  found  it 

among  the  decomposition  products  of  casein.  Its  presence  in  other 

7 8 

proteins  was  later  shown  hy  his  pupils  Ernst  Eischer  , Siegfried  , 

and  Hedin5.  Lysine  was  also  found  "by  Kutscher'1'0  in  antipeptone 

and  by  Kossel1"1"  in  the  protamines.  Its  occurrence  in  germinating 

12 

seedlings  was  demonstrated  by  Schulze  and  in  the  vegetable  pro- 

.13 


teins  by  Schulze  and  7/inters te in' 


Like  arginine  and  histidine, 


lysine  is  a very  widely  occurring  constituent  of  the  proteins. 

14 

Drechsel  gave  lysine  the  formula  an<i  regarded 

15 

it  as  a diamino  caproic  acid.  In  1899  Ellinger  proved  that  it 
possessed  this  constitution  by  obtaining  cadaverine  from  it  by  pu- 
trefaction. This  reaction  also  showed  that  the  two  amino  groups 
were  in  the  ex  and  e position. 

iTH2-CH2-CH2-CH2-CH2-9H-C00K *.C0g  + Mg-CH^CHg-CH^CHg-CHg-Mg 

- ITHg 

T 6 

Henderson's  experiments  also  showed  that  lysine  must  be  ct-e-di- 

amino  caproic  acid.  The  constitution  was  finally  definitely  deter- 

1 7 

mined  by  Eischer  and  V/eigert  by  the  following  synthesis:  ir-cyan- 
opropyl  malonic  ester  is  treated  with  nitrous  acid  and  loses  one  of 
its  carboxethyl  groups  and  is  converted  into  cx-oximido-^-cyano val- 
erianic ester,  which  on  reduction  with  sodium  amalgam  yields  <x-e-di- 
amino  caproic  acid.  The  reactions  are: 


n C-CEg-GHg-CHg-CH  , 


£00CoHc 

/ 


-V  NC-CH  -CHg-CHg-C-COOCgHg 


C00C2H5 


ITOH 


nh2-ce2-ce2-ch2-ch2-ch-coch 


* 


* 


' t 


-5- 


1 R 

Sorensen  also  accomplished  a synthesis  in  1903  hy  the  following 
series  of  reaction: 


»COOC  0H5 


fiQ* 


,C00C?Hp: 

= CcH„  N-CH  & + K3r 


C/-H/  UK  + BrCH  « ^ —v».n 

6 'co'  vcooc2h5  6xco  'cooc2h5 


C0V  ^.COOGpHp 

c Aa  h-ch 

6x#0'  KC00Co  H 
2 5 


+ G1-(CH2)3CU 


UaOCpHp-  CON  .C00CpHR 
K-CT-COOCpH? 

6^0/  *^(CH  JJs 

c o 


Na 


.COx  ^COOCpHp-  Con< 

> 0 PH/,  ii-g-cooc;h?  

° ( CH  f3CH  1IH  HC1 


+alcohol  'Cd 


* c2heoh  + co2+ c6h4 


+ HH„-CH„-CH„-CH9-CH.-CH-COOH 

C*  Cj  Uj  JT-r- 

2 


COOH 

COOH 


Neither  of  these  syntheses  were  found  to  he  very  suitable 

for  the  preparation  of  a good  yield  of  lysine.  A better  synthesis 

19 

was  described  by  J.  von  Braun  • in  1909.  Benzoyl  piperidine  is  con- 
verted by  the  action  of  phosphorus  pentachloride  into  benzoyl  amino 
amyl  chloride;  the  chlorine  group  is  replaced  by  the  CIT  group  which 
is  hydrolyzed  to  the  COOH  group.  The  resulting  e-benzoyl  amino 
caproic  acid  is  brominated  with  phosphorus  and  bromine.  Treatment 
with  ammonia  gives  e-benzoyl  amino -tf-amino  caproic  acid  from  which 
lysine  is  obtained  by  hydrolysis.  The  reactions  are: 


C6H5 


"°-;d> 


C6H5C°-f-CHE-CH2-CH2 

C H C0-UH-CH  -CH  -CH0 
6 5 1 2 2 2 

C H CO-UH-CH  -CH  -CH 
6 5 | 2 2 2 

C H CO-HH-CH  -CH  -CH_ 
6 5 t 2 2 2 

C H CO-UH-CH  -CH  -CH 

6 5 | 2 2 2 

C6H5C°-1TH-CH2-CH2-CH2 

NH2-CH2-CH2-CH2 


-ch2-ch2-ci 

-CH  -CH  -I 

2 2 

-CH  -CH  -CN 
2 2 

-CH  -CH  -COOH 

-CH  -CHBr-COOH 
2 

-ch2-chm2-cooh 

-CH  -C HUH  -COOH 

cZ,  cZ 


• / 


' "/  K. 


-6- 


The  above  synthesis  was  stated  to  he  very  satisfactory,  hut  the 

actual  yield  of  lysine  from  benzoyl  piperidine  was  not  stated. 

Of  the  above  syntheses,  the  latter  gave  most  promise  of 

success  and  was  adopted  for  the  purpose  of  this  investigation.  As 

no  henzoyl  piperidine  was  available,  a method  had  to  he  worked  out 

21 

for  the  preparation  of  this  compound.  In  1884  Ladenburg  obtained 
piperidine  by  the  action  of  sodium  on  pyridine  in  an  alcoholic  so- 
lution. The  author  states  that  the  reaction  goes  only  in  a small 
part,  and  no  improvements  have  appeared  in  the  literature  since  that 
time.  A method  for  the  reduction  of  pyridine  to  piperidine  by  means 
of  sodium  in  alcoholic  solution  was  perfected.  The  piperidine  was 
not  isolated,  but  the  crude  product  was  converted  into  the  benzoyl 
derivative.  By  development  of  technique  and  by  increasing  the  speed 
of  the  reaction  the  yield  was  increased  from  about  70$  to  85-90$. 

The  benzoyl  piperidine  obtained  by  vacuum  distillation 

was  treated  with  phosphorus  pentachloride  as  with  a few  exceptions 

20  1 9 

described  by  Braun  . Braun  suggests  that  a better  yield  of 
6-benzoyl  amino  amyl  cyanide  is  obtained  if  iodine  is  substituted 
for  chlorine  by  refluxing  the  alcoholic  solution  with  potassium  io- 
dide before  the  addition  of  the  sodium  cyanide.  This  was  confirmed 
by  experiment. 

6-Leucine  was  prepared  as  described  by  Braun22  in  1907, 
by  the  complete  hydrolysis  of  6-benzoyl  amino  amyl  cyanide  with  hy- 
drochloric acid.  The  amino  acid  was  freed  of  the  hydrochloric  acid 
by  treatment  with  silver  oxide  and  subsequent  treatment  with  hydro- 
gen sulfide  to  remove  the  excess  silver  from  solution.  This  com- 

23 

pound  has  also  been  prepared  by  Gabriel  and  Maas  by  the  complete 
hydrolysis  of  phthalimido  butyl  malonic  ester: 


< 


N 


w - 

. J , {,  . 


t 


-7- 


COOH 


+ 2 G2H5OH 


+ C02+  im2-CEp-CH2-GH2-CH2-CH2-C00H 


Upon  heating,  €-leucine  condenses  to  form  an  inner  amide 
according  to  the  reactions: 


ITHg(CHg)  5COOH 


I + KgO 


23 

Gabriel  and  Maas"  found  this  inner  amide  to  he  the  product  of  the 


vacuum  distillation  of  e-leucine.  By  hydrolysis  with  hydrochloric 
acid,  the  amide  is  converted  to  the  hydrochloride  of  the  amino  acid. 


zoyl  chloride,  and  the  resulting  e-benzoyl  amino  caproic  acid  was 
carefully  dried  and  brominated  with  bromine  and  phosphorus.  It  was 
found  important  to  have  all  of  the  reacting  substances  very  dry. 

€ -Benzoyl  amino -cub rom  caproic  acid  was  treated  with  ammonium  hy- 
droxide and  yielded  the  corresponding  e-benzoyl  amino -ae-amino  capro- 
ic acid  which  gave  on  hydrolysis  lysine  dihydrochloride. 


treated  with  calcium  carbonate  yielding  the  calcium  salt  of  6-ben- 
zoyl amino -oc-hy  dr  oxy  caproic  acid.  Treatment  with  hydrochloric 
acid  liberated  the  free  acid  which  gave  on  hydrolysis  the  hydro- 
chloride of  e-amino -<x-hydrozy  caproic  acid.  From  this  the  free 
amino  acid  was  prepared  by  the  ordinary  treatment  with  silver  oxide. 

Ct-Hydroxy  caproic  acid  was  obtained  from  a-brom  caproic 


£ -Leucine  was  benzoylated  in  alkaline  solution  with  ben 


6-Amino-oc-hydroxy  caproic  acid  was  prepared  by  the  method 
of  Fischer  and  3emplin^4.  6-Benzoyl  amino-a-brom  caproic  acid  was 


acid  by  boiling  it  with  a solution  of  sodium  carbonate.  The  acid 
was  isolated  as  the  copper  salt.  Upon  treating  the  copper  salt 


-8- 


with  hydrogen  sulfide,  filtering,  and  evaporating  under  diminished 
pressure,  the  free  hydroxy  caproic  acid  was  obtained. 

An  attempt  was  made  to  obtain  € -hydroxy  caproic  acid  from 
6-leucine.  The  latter  was  treated  with  nitrous  acid  at  a low  tem- 
perature. The  reaction  mixture  was  extracted  with  ether  and  the 
ether  evaporated  to  dryness.  A small  amount  of  a white  crystalline 
compound  with  a fruity  odor  was  obtained.  The  yield  was  very  poor, 
lack  of  time  prevented  further  investigation  of  this  and  other 
reactions • 


-9- 


EXPERIMEUTA1 

Benzoyl  piperidine 

A 5 liter  flask  is  placed  in  an  oil  bath  and  is  fitted 
with  a Y-tube  carrying  an  efficient  reflux  condenser.  3 liters 
of  absolute  alcohol  and  130  grams  of  pyridine  are  placed  in  the 
flask  and  1 lb.  of  sodium  is  added  through  the  Y-tube  in  10  gram 
portions  during  the  course  of  40  minutes.  The  sodium  is  added 
rather  slowly  at  first  but  may  be  added  more  rapidly  as  the  boil- 
ing point  of  the  liquid  increases,  about  one-half  of  the  total 
amount  being  added  during  the  last  15  minutes. 

Fifteen  hundred  cc.  more  of  absolute  alcohol  is  now  add- 
ed in  300  cc.  portions  at  5 minute  intervals.  Add  a few  small 
pieces  of  clay  plate,  apply  a flame  to  the  oil  bath,  and  reflux 
until  all  of  the  sodium  disappears.  A large  amount  of  melted 
sodium  will  be  present  in  the  bottom  of  the  flask  which  will  rise 
to  the  surface  on  refluxing  and  requires  2 or  3 hours  for  complete 
solution.  Replace  the  Y-tube  with  a stopper  carrying  a dropping 
funnel  and  condenser  arranged  for  downward  distillation.  As  the 
alcohol  is  distilled  off,  water  is  added  until  a total  of  1800  cc. 
has  been  added.  (Caution:  addition  of  water  causes  veiy  rapid 

distillation  of  alcohol.) 

The  distillation  is  carried  on  until  a total  of  about 
6 liters  has  been  distilled  over,  at  which  time  the  distillate 
will  appear  milky  and  the  flask  will  contain  only  a concentrated 
solution  of  NaOH  which  should  be  diluted  at  once  and  discarded. 

The  distillate  is  acidified  with  300  cc.  of  cone.  KC1  and  the 
alcohol  distilled  off  on  a steam  cone  leaving  behind  the  piperi- 
dine hydrochloride.  This  distillation  is  continued  until  a volume 


!. 


' ) 


* V- 


-10- 


of  about  600  cc.  remains  in  the  flask.  Transfer  the  solution  to  a 
2 liter  flask  using  E00  cc.  of  v/ater  to  wash  the  solution  complete- 
ly into  the  second  flask  which  is  now  fitted  with  a reflux  condens- 
er, dropping  funnel,  and  mechanical  stirrer  running  in  a mercury 
seal  • 

The  mixture  is  vigorously  stirred  and  a solution  of  176 
grams  of  NaOH  in  300  cc.  of  water  is  added  through  the  condenser. 
235  grams  of  benzoyl  chloride  is  run  in  through  the  dropping  fun- 
nel during  the  course  of  an  hour.  A pan  of  water  near  room  temper- 
ature is  placed  under  the  flask.  The  mixture  is  stirred  15  minutes 
longer  and  allowed  to  stand  for  a few  minutes,  whereupon  the  ben- 
soyl  piperidine  separates  as  a dark  layer  above  the  salt  solution. 
Pour  the  mixture  carefully  into  a separatory  funnel  and  draw  off 
the  aqueous  portion.  The  oily  layer  is  removed  to  a 500  cc. 

Claisen  distilling  flask  and  vacuum  distilled.  The  first  distil- 
late may  be  pink  in  color  but  becomes  a straw  color  upon  a second 
distillation.  Boiling  points: 

At  17  mm.  191°-194° 

At  130  mm.  240°-244° 

For  the  purposes  of  this  work  a single  distillation  was  found  to 
give  a sufficiently  pure  product.  A yield  of  E75-280  grams  or  87- 
89$  of  the  theory  was  obtained  in  several  consecutive  runs. 

Notes.  Unless  the  purity  of  the  pyridine  is  known,  it 
should  be  distilled  before  use.  For  this  work  a fraction  boiling 
at  112°-117°  was  used. 

A troublesome  emulsion  sometimes  results  after  benzoyla- 
tion.  This  may  usually  be  broken  up  by  the  addition  of  more 
strong  NaOH  solution.  Any  NaOH  carried  into  the  distilling  flask 


... 


-11- 


causes  decomposition  of  the  benzoyl  piperidine  and  consequently  a 
considerably  lower  yield.  For  this  reason  it  is  well  to  wash  the 
product  carefully  in  the  separatory  funnel  with  a little  water  after 
the  alkaline  solution  has  been  drawn  off.  In  case  an  emulsion  is 
formed  which  cannot  be  broken  up,  it  is  possible  to  extract  the 
product  with  benzene.  This  is  only  necessary  however  in  extreme 
cases • 

Benzoyl  piperidine  as  obtained  above  is  a straw  colored 
viscous  liquid.  Upon  long  standing  or  seeding  with  crystalline 
benzoyl  piperidine,  the  compound  crystallizes  in  long  colorless 
needles  which  melt  at  48°  C.  If  the  product  is  impure,  it  partial- 
ly crystallizes  to  a semi  solid  consistancy. 

It  may  be  noted  that  a large  excess  of  sodium  is  used. 

For  this  reason  it  seemed  possible  that  a larger  portion  of  pyri- 
dine might  be  reduced.  Accordingly  a run  was  made  using  195  grams 
of  pyridine  and  the  same  quantity  of  sodium  and  alcohol.  The  bases 
were  neutralized  with  300  cc . of  EC1,  later  made  alkaline  with  250 
grams  of  NaOH,  and  benzoylated  with  360  grams  of  benzoyl  chloride. 
Yield:  345-365  grams,  71-75$. 

Rapid  reduction  is  necessary,  the  limiting  factor  being 
the  efficiency  of  the  condenser.  By  shortening  the  time  allowed 
for  the  addition  of  the  sodium  from  one  hour  to  40  minutes,  the 
yield  was  increased  by  20$.  The  condenser  used  was  130  cm.  in 
length  and  had  a diameter  of  1.5  cm.  This  condenser  gave  consid- 
erable trouble  with  clogging  whereupon  relief  was  obtained  by  re- 
moving momentarily  the  stopper  in  the  Y-tube.  A condenser  of 
dimensions  2 X 180  cm.  is  recommended.  The  Y-tube  had  a diameter 
of  3 cm. 


. 


f 


. 

. 


-12- 

If  the  alcohol  solution  is  allowed  to  cool  before  the 
addition  of  the  water,  it  solidifies  and  is  only  remelted  with 
great  difficulty.  If  it  is  desired  to  suspend  the  process  the 
water  should  he  first  added  and  then  the  solution  will  not  solidi- 
fy* 

Benzoyl  piperidine  is  much  given  to  superheating  making 

distillation  difficult. 

20 

C-Benz oyl  amino  amyl  chloride 

Two  hundred  twenty  grams  of  good  quality  benzoyl  piperi- 
dine is  placed  in  a 1 liter  round  bottom  flask  together  with  240 
grams  of  C.P.  phosphorus  pentachloride . Attach  the  flask  to  a 
reflux  condenser  with  a tight  fitting  cork  and  warm  gently.  Have 
in  readiness  a pan  of  ice  water.  The  reaction  is  very  vigorous 
when  once  started  and  must  be  checked  by  application  of  the  cold 
water  to  the  flask,  in  order  to  keep  the  foam  from  being  carried' 
up  into  the  condenser.  The  mixture  melts  together  in  this  manner 
to  a cherry  red  liquid.  When  the  vigorous  reaction  has  ceased, 
reflux  with  a very  low  flame  for  2-§-  hours. 

How  cool  the  mixture  slightly  and  with  stirring  pour  it 
slowly  upon  a kilogram  of  ice.  A heavy  oil  separates  in  the  bottom 
of  the  beaker.  Stir  this  up  in  the  ice  water  in  order  to  remove 
as  much  of  the  free  hydrochloric  acid  as  possible  and  set  aside 
over  night.  Upon  standing  the  tar  changes  to  a more  crystalline 
mass.  Decant  the  clear  liquid,  leaving  the  product  in  the  beaker. 
Upon  breaking  this  up  with  a stirring  rod  it  is  found  to  be  strong- 
ly acid.  Place  the  beaker  containing  the  product  in  a pan  of  ice 
water  and  stir  into  the  latter,  a solution  of  strong  sodium  hydrox- 


. 


. 


* . ' v. 


-13- 

ide  until  the  liquid  "becomes  neutral.  In  this  way  the  free  acid 
is  completely  removed  from  the  product.  Dilute  to  1^-  liters,  heat 
the  neutral  mixture  just  to  boiling,  and  stir  vigorously  in  order 
to  thoroughly  wash  the  product.  Allow  the  mixture  to  cool.  Upon 
standing  the  6-benzoyl  amino  amyl  chloride  crystallizes  as  a solid 
cake  in  the  bottom  of  the  beaker  and  as  large  flakes  distributed 
throughout  the  supernatent  liquid.  Yield  about  170  grams  or  65$ 
of  the  theory.  For  the  purposes  of  this  investigation  it  was 
found  impractical  to  further  purify  the  compound.  It  may  be  re- 
crystallized from  mixtures  of  water  and  acetone  or  of  ethyl  ether 
and  petroleum  ether,  in  which  cases  the  compound  comes  down  in 
pure  white  flakes  which  melt  at  63°  C.  6-Benzoyl  amino  amyl 
chloride  is  easily  soluble  in  all  of  the  common  organic  solvents 
except  petroleum  ether.  J.  von  Braun2^  succeeded  in  distilling  the 
compound  in  vacuum  and  gave  its  boiling  point  as  230-240°  C.  at 
12  mm. 

Notes:  Hydrochloric  acid  is  evolved  during  the  reaction 

and  should  be  conducted  from  the  top  of  the  condenser  through  a 
calcium  chloride  tube  to  a beaker  of  water. 

It  was  found  necessary  to  use  C.P.  phosphorus  penta- 
chloride  as  the  commercial  product  reduced  the  yield  appreciably. 
According  to  Ruzicka  the  phosphorus  pentachloride  should  be  added 
in  four  portions,  the  reaction  mixture  being  heated  to  boiling  and 
cooled  between  successive  portions,  but  it  was  found  more  conven- 
ient and  efficient  to  add  it  all  at  the  beginning  and  to  control 
the  reaction  by  means  of  ice  water. 

The  time  of  boiling  the  reaction  mixture  was  reduced 
from  !-§■  hours  to  £ hour  with  the  result  that  the  yield  was  greatly 


-14- 


diminished.  2-|-  hours  seem  to  give  the  maxiumm  yield. 

In  the  development  of  the  synthesis  an  attempt  was  made 
to  purify  the  crude  6-benzoyl  amino  amyl  chloride  by  steam  distil- 
lation. By  steam  distillation  without  previous  careful  neutraliza- 
tion almost  the  entire  product  was  lost,  due  to  hydrolysis.  7/hen 
the  crude  product  was  carefully  neutralized  with  sodium  hydroxide 
and  sodium  carbonate  before  steam  distillation,  hydrolysis  did  not 
take  place  and  about  5 cc.  of  a light  oil  was  obtained,  probably 
pentamethylene  chloride.  This  method  was  abandoned  as  having  few 
advantages . 

6-Benzoyl  amino  amyl  cyanide^9 

Without  further  purification,  the  chlorine  compound  ob- 
tained in  the  above  preparation  is  dissolved  in  800  cc.  of  alcohol 
and  refluxed  on  the  steam  bath  with  160  grams  of  potassium  iodide 
for  about  10  hours.  The  mixture  is  cooled,  whereupon  the  insoluble 
salts  separate  out  and  are  filtered  off.  A solution  of  140  grams 
of  sodium  cyanide  in  300  cc.  of  water  is  now  added  and  the  mixture 
refluxed  once  more,  this  time  for  about  20  hours.  At  the  end  of 
this  time  the  condenser  is  arranged  for  downward  distillation  and 
the  alcohol  is  distilled  off  until  only  about  400  cc.  remains. 

This  residue  is  poured  into  a liter  of  cold  water  and  the  mixture 
thoroughly  stirred  up  to  wash  the  cyanide.  Upon  standing,  the 
6-benzoyl  amino  amyl  cyanide  solidifies,  partly  as  a solid  crystal- 
line cake  and  partly  as  almost  white  crystals  from  the  water-alco- 
hol liquid.  Yield  of  crude  product  is  about  equal  to  the  weight  of 
chloride  compound  used.  The  product  is  impure  and  the  actual  yield 
of  cyanide  is  difficult  to  determine.  This  product  is  directly 


-In- 
applicable for  hydrolysis  to  either  f'-benzoyl  amino  caproic  acid  or 
to  €-amino  caproic  acid.  For  purification  the  cyanide  may  he  re- 
crystallized from  30 jo  alcohol.  Melting  point:  95°  C. 

Notes:  J.  von  Braun1^  calls  attention  to  the  fact  that 

hy  going  from  the  chlorine  compound  to  the  cyanide  through  the 
corresponding  iodine  compound,  the  yield  is  much  better  than  in 
the  case  of  direct  substitution  of  cyanide  for  chlorine.  This  was 
confirmed  by  experiment. 

pp 

6-Amino  caproic  acid*'"'' 

One  hundred  grams  of  the  crude  e -benzoyl  amino  amyl  cya- 
nide is  placed  in  a 1 liter  flask  and  refluxed  with  500  cc . of  1 to 
1 hydrochloric  acid  for  9 hours.  The  mixture  is  then  cooled  and 
the  benzoic  acid  and  unhydrolyzed  residue  (about  E5  grams)  is  fil- 
tered off.  The  filtrate  is  evaporated  to  dryness  on  the  water 
bath.  The  residue  is  brown  in  color  and  consists  of  the  hydro- 
chloride of  6-amino  caproic  acid  and  some  inorganic  salts  as  im- 
purities. The  residue  is  taken  up  in  300  cc.  of  absolute  alcohol 
and  filtered  from  the  inorganic  salts.  The  alcoholic  filtrate  is 
evaporated  to  a paste.  The  dish  containing  the  hydrochloride  is 
placed  in  an  ice  bath  and  a mixture  of  50  grams  of  silver  oxide 
and  50  cc.  of  water  is  stirred  into  it.  After  about  10  minutes 
the  mixture  is  diluted  with  water  to  250  cc.  and  filtered.  Hydro- 
gen sulfide  is  passed  into  the  cold  solution  until  a portion  of 
the  filtrate  no  longer  gives  a test  for  silver  with  hydrochloric 
acid.  This  will  require  from  3 to  4 hours  at  room  temperature. 

The  silver  sulfide  precipitate  is  filtered  off  and  the  filtrate 
vacuum  distilled  at  a temperature  of  45°.  The  distillation  is  con- 


-16- 


time  & until  the  amino  acid  crystallizes  as  a soft  crystalline 
mass.  It  is  now  washed  out  of  the  distilling  flask  and  triturated 
with  absolute  alcohol,  decanted  through  a filter,  fresh  portions 
of  absolute  alcohol  added,  and  the  process  repeated  until  the 
6-amino  caproic  acid  becomes  pure  white  and  very  finely  divided. 
Finally  it  is  washed  into  the  filter  and  dried.  Yield:  12  grams 
or  20$  from  the  crude  cyanide. 

€ -Amino  caproic  acid  is  a white,  finely  crystalline  com- 
pound. It  dissolves  easily  in  water  but  is  insoluble  in  ether  and 
absolute  alcohol.  At  203°  C.  it  melts  to  a colorless  liquid. 

Notes:  The  unhydrolyzed  residue  may  be  collected  and 

again  refluxed  with  hydrochloric  acid. 

The  alcoholic  solution  of  the  hydrochloride  of  the  amino 
acid  is  deeply  colored.  Bone-black  was  used  in  an  attempt  to  de- 
colorize this  solution  but  seemed  to  have  little  effect. 

When  heated  € -leucine  readily  forms  an  inner  amide. 

When  the  solution  of  the  free  amino  acid  was  evaporated  in  the  or- 
dinary way  on  the  steam  cone,  the  yield  of  the  amino  acid  was 
greatly  diminished,  due  to  the  formation  of  this  amide.  Even  in 
the  case  of  vacuum  distillation,  the  yield  of  the  inner  amide  was 
slightly  greater  than  that  of  the  free  amino  acid.  This  amide  is 
very  soluble  in  alcohol  and  is  removed  by  extraction  of  the  amino 
acid  with  absolute  alcohol.  Upon  concentration  of  the  alcoholic 
extract  and  subsequent  hydrolysis  with  hydrochloric  acid,  the  in- 
ner amide  was  converted  quantitatively  into  the  hydrochloride  of 
the  amino  acid. 

Litharge  was  used  in  an  attempt  to  free  the  amino  acid 
from  the  hydrochloride,  but  complete  removal  of  the  chlorine  could 


-17- 


not  be  obtained  by  this  method. 

e -Benzoyl  amino  caproic  acid4^ 

One  hundred  grams  of  the  crude  e-benzoyl  amino  amyl  cya- 
nide is  completely  hydrolyzed  with  hydrochloric  acid  as  in  the 
preparation  of  e-amino  caproic  acid.  The  product  is  dissolved  in 
alcohol  as  before  and  filtered  from  the  insoluble  inorganic  salts. 
The  hydrochloride  obtained  by  the  evaporation  of  the  alcohol  is 
taken  up  in  a solution  of  400  cc.  of  water  and  100  grams  of  sodium 
hydroxide.  The  mixture  is  cooled  and  placed  in  a flask  equipped 
with  a mechanical  stirrer.  50  grams  of  benzoyl  chloride  is  run  in 
with  stirring  during  the  course  of  30  minutes.  Stirring  is  contin- 
ued 15  minutes  longer.  Filter  with  suction  to  remove  any  benzamide 
which  has  been  formed.  The  solution  is  acidified,  whereupon  the 
6-benzoyl  amino  caproic  acid  separates  as  a heavy  oil.  Upon  stand- 
ing over  night  this  changes  to  a brown,  oily,  crystalline  mass. 

The  mother  liquor  is  decanted  and  the  acid  is  dried  bet ween  clay 
plates  in  a vacuum  dessicator.  The  product  is  now  applicable  for 
bromination.  Yield  from  100  grams  of  cyanide  45  grams,  41$  of  the 
theory.  6-Benzoyl  amino  caproic  acid  is  easily  soluble  in  ether 
or  alcohol  and  insoluble  in  petroleum  ether.  It  may  be  recrystal- 
lized from  ether  and  alcohol.  The  compound  crystallizes  in  long 
needles  which  mat  together  in  a sticky  mass.  Melting  point:  79°  C. 

Notes:  By  boiling  the  cyanide  with  50  grams  of  sodium 

hydroxide,  £00  cc.  of  water,  and  500  cc.  of  alcohol  for  3 hours 
the  cyanide  may  be  saponified  without  effect  upon  the  benzoyl  radi- 
cal. The  product  is  very  difficult  to  separate  from  the  reaction 
mixture  and  contains  a great  many  impurities  from  the  crude  cyanide 


-18- 


which  cannot  be  removed.  Both  methods  were  thoroughly  investigat- 
ed and  it  was  found  more  practicable  to  completely  hydrolyze  and 
benzoylate  bach  as  described  above. 

In  some  cases  a crystalline  product  was  not  formed  even 
after  standing  several  days.  The  cause  of  this  failure  was  not 
determined.  An  attempt  was  made  to  benzoylate  the  6-amino  caproic 
acid  without  first  removing  the  inorganic  salts.  In  this  case  a 
large  amount  of  benzamide  and  benzoic  acid  was  formed  and  the 
yield  was  considerably  reduced. 

6-Benzoyl  amino-oubrom  caproic  acid 

Forty  grams  of  C -benzoyl  amino  caproic  acid  is  very 
thoroughly  dried  over  sulfuric  acid  in  a vacuum  dessicator.  The 
dry  acid  is  mixed  in  a mortar  with  5 grams  of  red  phosphorus  which 
has  also  been  previously  dried.  The  red  viscous  mass  is  scraped 
into  a 500  cc . flash  equipped  with  a rubber  stopper  bearing  a 
calcium  chloride  tube  and  a small  dropping  funnel.  Forty  cc.  of 
bromine,  which  has  been  dried  under  sulfuric  acid,  is  placed  in 
the  funnel  and  allowed  to  drop  slowly  upon  the  reaction  mixture. 
The  first  drops  of  bromine  react  violently  with  the  appearance  of 
fire.  After  the  first  few  cc.  have  been  added,  the  remainder  of 
the  bromine  is  allowed  to  flow  at  the  rate  of  about  60  drops  per 
minute.  The  reaction  mixture  is  hept  in  a plastic  state  by  cool- 
ing the  flash  in  a dish  of  cold  water. 

After  all  of  the  bromine  has  been  added,  the  calcium 
chloride  tube  and  dropping  funnel  are  replaced  by  an  air  condenser 
closed  at  the  upper  end  by  a calcium  chloride  tube.  The  flash  is 
now  heated  on  the  steam  bath  until  the  evolution  of  hydrobromic 


-19- 


acid  is  complete.  This  will  require  about  12  hours.  The  mixture 
is  poured  slowly  and  with  stirring  into  500  cc.  of  ice  water.  As 
the  acid  chloride  is  decomposed,  the  free  acid  separates  as  a 
brown  solid  mass.  Sulfur  dioxide  is  now  passed  into  the  brown 
solution  just  until  no  more  bromine  color  is  apparent.  The  mass 
of  acid  in  the  bottom  of  the  beaker  must  be  thoroughly  broken  up 
by  stirring  in  order  to  remove  the  excess  bromine.  The  crumbly, 
brown  mass  is  allowed  to  dry,  triturated  with  50  cc.  of  ethyl 
ether,  and  filtered  with  aiction.  It  may  be  washed  with  a few 
cc.  of  ether  to  remove  any  unchanged  acid.  Yield:  20  grams,  37$ 

of  the  theory.  6-Benzoyl  amino -cx-brom  caproic  acid  is  insoluble 
in  water,  easily  soluble  in  hot  alcohol,  and  only  slightly  solu- 
ble in  ether.  It  may  be  purified  by  recrystallization  from  dilute 
alcohol.  Melting  point:  166°  0.  The  brom  acid  obtained  by  this 

method  may  be  red  in  color  when  dry  due  to  incomplete  removal  of 
the  bromine. 

Ilotes:  The  reactive  substances  must  be  very  thoroughly 

dried  in  order  to  prevent  the  premature  decomposition  of  the  acid 
chloride . 

Caution  must  be  used  in  pouring  the  reaction  mixture 
into  water  as  the  decomposition  of  the  acid  chloride  is  accompan- 
ied by  the  evolution  of  heat,  and  the  reaction  is  very  vigorous 
if  the  water  becomes  hot.  It  should  be  kept  cool  and  allowed  to 
progress  slowly. 

J.  von  Braun  recommends  that  the  crude  acid  be  dissolved 
in  alcohol  after  decolorising  with  sulfur  dioxide.  After  fil- 
tration of  the  alcoholic  solution  the  acid  is  precipitated  by 
addition  of  water.  This  was  found  to  be  unnecessary. 


. 0 


l 


..  . . - V ' 


N 


‘ 'C 


:i 


! 


-20- 


€ -Benzoyl  lysine 

Thirty  grams  of  6-benzoyl  amino-a-brom  caproic  acid  is 
placed  in  a strong,  well  stoppered  bottle  with  300  cc.  of 
ammonium  hydroxide.  The  bottle  is  put  in  a warm  place  and  held 
at  30°-40°  for  two  days.  The  contents  are  filtered,  any  residue 
being  washed  with  two  portions  each  of  200  cc.  of  hot  water.  If 
solution  of  the  product  does  not  appear  to  be  complete,  more  hot 
water  may  be  used.  The  combined  filtrates  are  evaporated  to  dry- 
ness upon  the  steam  bath  whereupon  the  product  separates  in  large 
white  flakes.  The  residue  is  triturated  with  75  cc.  of  absolute 
alcohol,  filtered,  and  washed  into  the  filter  with  small  portions 
of  absolute  alcohol.  Yield  about  12  grams  or  50%  of  the  theory. 
e-Benzoyl  lysine  is  insoluble  in  alcohol  and  ether  and  is  only 
slightly  soluble  in  v/ater.  It  melts  at  265°  C. 

dl -lysine  dihydrochloride 

Twelve  grams  of  6-benzoyl  lysine  and  50  cc.  of  cone, 
hydrochloric  acid  is  placed  in  a thick  walled  glass  tube  and  the 
tube  is  sealed  off.  The  reaction  mixture  is  heated  at  115°-125° 
in  an  oil  bath  for  5 hours.  At  the  end  of  this  time  the  tube  is 
cooled  and  opened  and  the  acid  diluted  with  75  cc . of  v/ater. 

The  separated  benzoic  acid  is  filtered  off  and  the  filtrate  ex- 
tracted twice  with  40  cc . of  ether. 

The  acid  solution  is  then  boiled  with  2 grams  of  bone- 
black  ( "Norite")  for  5 minutes  and  filtered.  The  filtrate  is 
evaporated  on  the  steam  hath  to  a light  yellow  syrup.  This  is 
taken  up  in  100  cc.  of  absolute  alcohol  and  filtered.  Upon  cool- 
ing and  adding  an  equal  volume  of  dry  ether,  the  hydrochloride 


-El- 


separates  as  a sticky  mass  in  the  bottom  of  the  beaker.  After  the 
liquid  has  become  clear,  decant  the  alcohol-ether  mixture  and  dry 
the  hydrochloride  in  the  beaker  in  a dessicator.  After  drying  for 
2 or  3 days,  the  lysine  hydrochloride  may  be  scraped  from  the 
beaker.  The  yield  from  12  grams  of  benzoyl  lysine  is  5 grams  or 
about  47 % of  the  theoretical  amount,  lysine  hydrochloride  readily 
absorbs  moisture  from  the  air  and  must  be  kept  in  a tightly  stop- 
pered bottle.  It  is  very  soluble  in  water  and  alcohol  but  insolu- 
ble in  ether.  It  melts  v/ith  decomposition  at  192°-193°  C. 

Calcium  salt  of  6-benzoyl  amino -oc-hydroxy  caproic  acid 

Thirty  grams  of  6-benzoyl  amino-d-brorn  caproic  acid  is 
dissolved  in  375  cc.  of  hot  Q0%  alcohol,  and  10  grams  of  precipi- 
tated calcium  carbonate  is  stirred  into  the  solution  whereupon 
strong  evolution  of  carbon  dioxide  takes  place.  The  turbid  sus- 
pension thus  formed  is  poured  slowly  into  3-g-  liters  of  boiling 
water,  and  15  grams  of  calcium  carbonate  is  again  added.  The  mix- 
ture is  boiled  15  minutes  longer,  then  filtered,  and  the  liquid 
evaporated  under  diminished  pressure  to  about  250  cc.  The  calcium 
salt  of  €-benzoyl  amino - ct-hy dr oxy  caproic  acid  separates  as  a fine, 
crystalline,  powder  which  is  filtered  from  the  yellow  mother 
liquor.  The  yield  is  20  grams  or  about  72%  of  the  theory.  The 
product  is  of  a light  yellow  color. 

P4- 

e- Amino -Ct-hy  dr  oxy  caproic  acid 
Thirty  grams  of  the  calcium  salt  of  6-benzoyl  amino- 
OC-hydroxy  caproic  acid  as  prepared  above  is  treated  with  25  cc.  of 
5-normal  hydrochloric  acid  which  is  then  diluted  with  water  to  a 
volume  of  60  cc.  The  salt  slowly  goes  into  solution  and  a heavy. 


-22- 


reddish  brown  oil  separates  in  the  "bottom  of  the  flask.  The  acid 
solution  is  cooled  to  C°  C.  and  decanted. 

The  oil  remaining  in  the  flask  is  dissolved  in  200  cc.  of 
5-normal  hydrochloric  acid  and  refluxed  for  5 hours.  Upon  cooling, 
"benzoic  acid  crystallises  out  and  is  separated  "by  filtration.  The 
acid  solution  is  then  extracted  3 times  with  50  cc.  of  ethyl  ether 
to  remove  the  last  traces  of  "benzoic  acid  from  solution,  and  is  then 
evaporated  under  diminished  pressure  to  a thick  syrup.  The  syrupy 
residue  is  dissolved  in  150  cc.  of  water  and  treated  with  25  grams 
of  silver  oxide.  After  about  10  minutes  the  liquid  is  filtered.  A 
portion  of  the  filtrate  should  give  no  turbidity  with  a drop  of 
silver  nitrate  solution.  The  filtrate  is  now  saturated  with  hydro- 
gen sulfide  to  precipitate  any  dissolved  silver  and  the  the  solu- 
tion is  again  filtered.  The  final  filtrate  is  evaporated  almost  to 
dryness  under  diminished  pressure.  The  €-amino-o£-hydroxy  caproic 
acid  separates  as  microscopic,  white  crystalls,  which  are  triturated 
with  absolute  alcohol,  filtered,  and  washed  to  whiteness  with  alco- 
hol. Yield:  8 grams  or  about  50%.  The  acid  melts  with  decomposi- 

tion and  a brown  coloration  at  220u-225°. 

pA 

Notes:  Uischer  and  Zemplin  isolated  the  6-benzoyl 

amino -tt-hydroxy  caproic  acid.  Upon  standing  in  a cold  place  the 
brown  oil  described  above  changes  to  a crystalline  solid  which  may 
be  recrystallized  from  boiling  water.  It  dissolves  easily  in  alco- 
hol and  acetone  and  with  difficulty  in  ether.  The  melting'  point 
was  given  as  107°  0. 

The  above  authors  used  hydrochloric  acid  to  remove  the 
excess  silver  from  solution  but  it  was  found  more  satisfactory  to 
use  hydrogen  sulfide  instead. 


-23- 

The  final  solution  for  evaporation  is  of  a straw  yellow 
color.  Bone-black  was  used  in  an  attempt  to  remove  this  color 
but  seemed  to  have  little  effect.  During  evaporation  the  color  be- 
comes intensified.  By  washing  the  product  with  absolute  alcohol, 
practically  all  of  this  coloring  matter  is  removed. 

Copper  salt  of  <X-hydroxy  caproic  acid 

Seventy  grams  of  a-brom  caproic  acid  was  dissolved  in  a 
solution  of  70  grams  of  sodium  carbonate  in  a liter  of  water.  The 
solution  was  boiled  thirty  minutes,  cooled,  and  filtered  to  remove 
a small  amount  of  a waxy  residue  from  the  (X-brom  caproic  acid.  The 
filtrate  was  again  heated  to  boiling,  and  35  grams  of  copper  acetate 
was  added  and  stirred  until  completely  dissolved.  The  copper  salt 
of  cx-hyaroxy  caproic  acid  precipitates  as  a light  colored  scum. 

The  solution  is  allowed  to  cool  and  crystallize  out  and  the  copper 
salt  is  filtered  off  and  washed  on  the  filter  with  cold  water.  It 
is  of  a light  blue-green  color.  Yield:  40  grams  or  60$  of  the 

theory. 

(X-Eydroxy  caproic  acid 

Twenty  grams  of  the  copper  salt  of  d-hydroxy  caproic  acid 
was  placed  in  a 1 liter  flask  and  shaken  with  800  cc.  of  boiling 
water  until  all  of  the  salt  was  in  suspension  or  in  solution.  The 
liquid  was  now  kept  hot  and  saturated  with  hydrogen  sulfide  for 
5 hours.  A colloidal  solution  appeared  to  have  been  formed.  This 
was  broken  up  by  allowing  the  liquid  to  stand  several  days  after 
which  it  was  filtered.  The  filtrate  and  washings  were  evaporated 
under  diminished  pressure  to  a syrup.  Upon  cooling,  this  syrup 


-25- 

SUMMARY 

1.  An  improved  method  for  the  preparation  of  benzoyl 
piperidine  has  been  developed. 

2.  Benzoyl  piperidine  has  been  converted  into  6-amino 
caproic  acid,  e-amino -cx-hydroxy  caproic  acid,  and  into  dl -lysine 
dihydrochloride  by  reactions  previously  studied  by  J.  von  Braun 
and  by  E.  Fischer  and  Zemplin. 

3.  <x-Hyaroxy  caproic  acid  has  been  prepared  from  the  cor- 
responding brom  compound. 

4.  6-Hydroxy  caproic  acid  could  not  be  obtained  in  reason 
able  yields  from  e-amino  caproic  acid  by  the  action  of  nitrous 

acid. 

5.  Sufficient  quantities  of  €-amino  caproic  acid,  cX-hy- 
droxy  caproic  acid,  dl-lysine  dihydrochloride,  and  e-amino-Qf-hy- 
droxy  caproic  acid  have  been  prepared  for  the  study  of  their  be- 
havior in  metabolism  experiments. 


-26- 


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